6-hydroxy-2,5,7,8-tetramethylchroman-compounds for the treatment of chronic obstructive airway diseases

ABSTRACT

Specifically, the present invention relates to (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl) (piperazin-1-yl)methanone or N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide or a pharmaceutically acceptable salt or base thereof for use in the treatment of chronic obstructive airway diseases, preferably chronic obstructive pulmonary disease (COPD) or asthma or bronchiectasis, more preferably chronic obstructive pulmonary disease (COPD).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/318,635, filed on Dec. 13, 2016, which is a 371 of PCT applicationnumber PCT/EP2015/063579 filed on Jun. 17, 2015, which claims priorityfrom NL application number 2013012, filed on Jun. 17, 2014. Allapplications are hereby incorporated by reference in their entireties.

The present invention relates to compounds for the treatment of chronicobstructive airway diseases such as chronic obstructive pulmonarydisease (COPD) or asthma or bronchiectasis. The present inventionfurther relates to drug delivery devices suitable to be used in thetreatment of chronic obstructive airway diseases such as a nebulizercomprising the present compounds.

Chronic obstructive pulmonary disease (COPD), also designated as chronicobstructive lung disease (COLD) or chronic obstructive airway disease(COAD) is a type of obstructive lung disease characterised by chronicobstruction of the airflow in the lungs. The main symptoms of chronicobstructive pulmonary disease (COPD) include shortness of breath, cough,and sputum production.

Tobacco smoking is the most common cause of chronic obstructivepulmonary disease (COPD) but also other causative factor's are knownsuch as air pollution and genetics.

Chronic obstructive pulmonary disease (COPD) can be prevented byreducing exposure to the known causes. This includes efforts to decreaserates of smoking and to improve indoor and outdoor air quality. Chronicobstructive pulmonary disease (COPD) treatments include: quittingsmoking, vaccinations, rehabilitation, and often inhaled bronchodilatorsand steroids. Some people may benefit from long-term oxygen therapy orlung transplantation.

Worldwide, chronic obstructive pulmonary disease (COPD) affects 329million people or nearly 5% of the population. In 2011, it ranked as thefourth-leading cause of death, killing 3 million people. The number ofdeaths is projected to increase due to higher smoking rates and an agingpopulation in many countries.

Asthma is common chronic inflammatory disease of the airwayscharacterized by variable and recurring symptoms, reversible airflowobstruction and bronchospasm. Common symptoms include wheezing,coughing, chest tightness, and shortness of breath.

Asthma is thought to be caused by a combination of genetic andenvironmental factors. Its diagnosis is usually based on the pattern ofsymptoms, response to therapy over time and spirometry. Asthma isclinically classified according to the frequency of symptoms, forcedexpiratory volume in one second (FEV1), and peal expiratory flow rate.Asthma may also be classified as atopic (extrinsic) or non-atopic(intrinsic) where atopy refers to a predisposition toward developingtype 1 hypersensitivity reactions.

Treatment of acute symptoms is usually with an inhaled short-actingbeta-2 agonist (such as salbutamol) and oral corticostereroids. In verysevere cases, intravenous corticosteroids, magnesium sulfate, andhospitalization may be required. Symptoms can be prevented by avoidingtriggers, such as allergens and irritants, and by the use of inhaledcorticosteroids. Long-acting beta agonists (LABA) or leukotrieneantagonists may be used in addition to inhaled corticosteroids if asthmasymptoms remain uncontrolled.

The occurrence of asthma has increased significantly since the 1970s. In2011, 235-300 million people globally have been diagnosed with asthmaand the number of deaths wherein asthma is the causative factor isestimated to be 250,000 deaths annually.

Bronchiectasis is a disease characterized by localized, irreversibledilation of part of the bronchial tree caused by the break down of themuscle and elastic tissue. It is classified as an obstructive lungdisease, along with emphysema, bronchitis, and asthma.

Involved bronchi are dilated, inflamed, and easily collapsible,resulting in airway obstruction and impaired clearance of secretions.Bronchiectasis may result from a variety of infective and acquiredcauses, including severe and recurrent pneumonia, tuberculosis, andcystic fibrosis. Bronchiectasis has both congenital and acquired causes,with the latter more frequent.

Tuberculosis, pneumonia, inhaled foreign bodies, allergicbronchopulmonary aspergillosis and bronchial tumours are the majoracquired causes of Bronchiectasis. Infective acquired causes associatedwith Bronchiectasis include infections caused by the Staphylococcus,Klebsiella, or Bordetella pertussis. Further, aspiration of ammonia andother toxic gases, pulmonary aspiration, alcoholism, heroin (drug use)and various allergies appear to be linked to the development ofBronchiectasis.

Bronchiectasis may also result from congenital causes that affect ciliamotility or ion transport. Kartagener syndrome is one such disorder ofcilia motility linked to the development of bronchiectasis. Anothercommon cause is cystic fibrosis affecting chloride ion transport.Young's syndrome, which is clinically similar to cystic fibrosis, isthought to significantly contribute to the development ofbronchiectasis. This is due to the occurrence of chronic infections ofthe sinuses and bronchiole tree. Other less-common congenital causesinclude primary immunodeficiencies, due to the weakened or nonexistentimmune system response to severe, recurrent infections that commonlyaffect the lung.

Chronic obstructive airway diseases, such as asthma, chronic obstructivepulmonary disease (COPD) and bronchodilators, including ß2-adrenergicreceptor (ß2-AR) agonists and anti-inflammatory agents likecorticosteroids, ß2-agonists are not effective as anti-inflammatorydrugs in vivo. Ideally, a drug has both bronchodilating andanti-inflammatory actions, without a risk of desensitization.Irrespective of a specific mode of action, preferably, a drug lowers theconstriction and, or lowers the inflammation, which has a positiveeffect of the efficacy of the lungs.

It is an object of the present invention, amongst other objects, toprovide compounds for the treatment of chronic obstructive airwaydiseases, such as asthma, chronic obstructive pulmonary disease (COPD)and bronchiectasis and especially chronic obstructive pulmonary disease(COPD) or asthma. In providing such compounds, the compound preferablymeet one or more of the modes of action for the medicament for thetreatment of chronic obstructive airway disease, i.e. compounds havingbronchodilating and anti-inflammatory effects. Preferably, the compoundsremain active over long term administration, i.e., the compounds showlittle desensitization.

The above object, amongst other objects, is met by the present inventionby as outlined in the appended claims.

The above object is met by a compound according to formula (I), or apharmaceutically acceptable salt or base thereof, for use in thetreatment of chronic obstructive airway diseases,

-   -   wherein R2 and R2 may be the same or different, and represent a        C1-C4 linear or branched alkylgroup;    -   wherein R3 represents a hydrogen or prodrug moiety that can be        removed in living tissue; preferably, R3 forms together with the        6-oxygen as ester group. R3 may have 1-12 carbon atoms,        preferably 1-6 carbon atoms, and may comprise one or more amine        or oxygen atoms;    -   n may be 0 or 1, and is preferably 1;    -   R4 is a group comprising at 1-20 carbon atoms and at least one        nitrogen atom; R4 may comprise further nitrogen atoms, one or        more oxygen atoms, halogen, sulphur or phosphor atoms and R4 may        comprise aromatic groups, wherein the molecular weight of R4        preferably is less than 300 Da;        wherein the compound is in formulation suitable for inhalation.

As will be recognized, the compound of formula (I) is derived fromtrolox, a water soluble analogue of vitamin E. In trolox, R1 and R2 aremethyl, R3 is hydrogen, and R4 is carboxylic acid.

Specifically, the above object, amongst other objects, is met by presentinvention by a compound according to the formula (II)

(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanone(II)

or a pharmaceutically acceptable salt or base thereof for use in thetreatment of chronic obstructive airway diseases, preferably chronicobstructive pulmonary disease (COPD) or asthma or bronchiectasis, morepreferably chronic obstructive pulmonary disease (COPD).

According to the present invention, according to a further aspect, theabove object, amongst other objects, are met by a compound according tothe formula (III)

N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide (III)

or a pharmaceutically acceptable salt or base thereof for use in thetreatment of chronic obstructive airway diseases, preferably chronicobstructive pulmonary disease (COPD) or asthma or bronchiectasis, morepreferably chronic obstructive pulmonary disease (COPD).

According to the present invention, according to a further aspect, theabove object, amongst other objects, are met by a compound selected fromthe group, together “group A”, consisting of2,2,5,7,8-pentamethylchroman-6-ol;(S)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid;(R)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid;6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;N-butyl-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;6-hydroxy-N-isopropyl-2,5,7,8-tetramethylchroman-2-carboxamide;(E)-N-(3,7-dimethylocta-2,6-dien-1-yl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(morpholino)methanone;N-(4-fluorobenzyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;6-hydroxy-N—((S)-2-hydroxy-1-phenylethyl)-2,5,7,8-tetramethylchroman-2-carboxamide;6-hydroxy-2,5,7,8-tetramethyl-N-(2-(methylamino)ethyl)chroman-2-carboxamide;6-hydroxy-2,5,7,8-pentamethyl-N-(2-(methylamino)ethyl)chroman-2-carboxamide;6-hydroxy-2,5,7,8-tetramethyl-N-(3-piperidin-1-yl)propyl)chroman-2-carboxamide;6-hydroxy-2,5,7,8-tetramethyl-N-(3-nitrophenyl)chroman-2-carboxamide;N-(4-fluorophenyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;methyl 4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide)benzoate;(4-butylpiperazin-1-yl)(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)methanone;(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone;((2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl)(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)methanone;N—((R)-2-amino-2-oxo-1-phenylethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone;N-(2-bromoethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;N′-(2-cyanoethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carbohydrazide;2-(((4-fluorobenzyl)amino)methyl)-2,5,7,8-tetramethylchroman-6-ol;2-((butylamino)methyl)-2,5,7,8-tetramethylchroman-6-ol;6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylic acid;2-(hydroxylmethyl)-5,7-diisopropyl-2,8-dimethylchroman-6-ol;6-hydroxy-N—((R)-1-hydroxypropan-2-yl)-2,5,7,8-tetramethylchroman-2-carboxamide;(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-(2-hydroxyethoxy)ethyl)piperazin-1-yl)methanone;N-(2-cyanoethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;6-hydroxy-N-(2-((2-hydroxyethyl)(methyl)amino)ethyl)-2,5,7,8-tetramethylchroman-2-carboxamide;(R)—N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;(S)—N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;2-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol;2-((((S)-2-hydroxy-1-phenylethyl)amino)methyl)-2,5,7,8-tetramethylchroman-6-ol;2,5,7,8-tetramethyl-2-(piperidin-1-ylmethyl)chroman-6-ol;N,6-dihydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxamide;(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone;(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone;2-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol;2-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol;2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)aceticacid;(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone;2-(4-(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carbonyl)piperazin-1-yl)aceticacid; ethyl2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetate;(S)-2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)aceticacid;(R)-2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)aceticacid;(2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylicacid;(2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylicacid;(2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylicacid and pharmaceutically acceptable salts or bases thereof for use inthe treatment of chronic obstructive airway diseases, preferably chronicobstructive pulmonary disease (COPD) or asthma or bronchiectasis, morepreferably chronic obstructive pulmonary disease (COPD).

The present inventors surprisingly discovered that the present compoundsaccording to formula (I), and most preferably(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanone orN,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide have anapparently bronchodilating and anti-inflammatory effect making themsuitable for the treatment of obstructive airways diseases and,especially, making them suitable for the treatment of chronicobstructive pulmonary disease (COPD) and asthma.

According to a preferred embodiment of the present invention, thepresent treatment of chronic obstructive airway diseases compromisesadministration of the present compounds such as the compounds accordingformula (I), (II), and (III), or according to group A, throughinhalation. Inhalation as used herein indicates a route ofadministration where the present compounds are taken in through themouth or nose, to arrive into the lungs.

The compound according to formula (I),

preferably has the following characteristics:

R1 and R2 may be the same or different, and represent a C1-C4 linear orbranched alkylgroup. Preferably, R1 and R2 are methyl, ethyl orisopropyl, and more preferably, R1 and R3 are the same, and are methylor isopropyl. Other suitable groups are n-butyl and t-butyl.

R3 represents a hydrogen or prodrug moiety that can be removed in livingtissue. Preferably, R3 forms together with the 6-oxygen an ester group.R3 may have 1-12 carbon atoms, preferably 1-6 carbon atoms, and maycomprise one or more amine or oxygen atoms. Suitable groups—togetherwith the 6-oxygen—include ethyl-ester, butyl-ester, benzoyl-ester, or anester of an amino-acid or amino acids wherein the aminogroup is amidatedwith an alkyl carboxylic acid having 1-4 carbon atoms. In one preferredembodiment, R3 is hydrogen.

n may be 0 or 1, and is preferably 1;

R4 is a group comprising at 1-20 carbon atoms at least one nitrogen atomR4 may comprise further nitrogen atoms, one or more oxygen atoms,halogen, sulphur or phosphor atoms and R4 may coarse aromatic groups.

The molecular weight of R4 preferably is less than 300 Da.

Preferably, the compound according to formula (I) has a molecular weightlower than 500 Da.

Preferably, the compound according to formula (I) does not comprise anaromatic heterocyclic ring.

Preferably, R4 comprises a carbonyl group, and most preferably, acarbonyl group attached to the trolox moiety.

In one preferred embodiment, R4 is —CO—N—R5, wherein the C═O is bound tothe trolox moiety, and wherein R5 is an alkylgroup, optionallysubstituted with nitrogen or oxygen wherein the alkylgroup comprises1-12 carbon atoms, and wherein nitrogen can be amine, quaternary amine,guanidine or imine, and oxygen can be hydroxyl, carbonyl or carboxylicacid. Oxygen and nitrogen together may form amide, urea or carbamategroups.

The alkylgroup in R5 may be linear, branched or cyclic, and preferablycomprises at least one cyclic structure.

Compounds as presented by formula (I) can be made according to knownchemical synthesis.

For example, compounds with a guanidine group, or a piperazine groupattached to a trolox moiety via an alkyl group are described inEP202580. Analogous synthesis can be used, wherein the 6-oxygen isprotected, and liberated after the synthesis, or protected with aprodrug-moiety.

For example, compounds with nicotinate groups as substituents, aredescribed in U.S. Pat. No. 4,618,890. The nicotinate attached to the6-oxygen of the trolox moiety can act as a prodrug moiety, which ishydrolysed in vivo to a free hydroxylgroup.

For example, suitable compounds are described in WO88/08424, examples18-23 and 778-164.

For example, suitable compounds are described in WO97/41121, inpreparations 1, 6, 7, 12-15, 21, 24 and 27, wherein the benzoylgroup canbe removed, or can act as a prodrug moiety.

Further compounds are described in e.g. WO03/024943, like compounds9-11, 25-28, 109-112, 119-122 etc.

For example, compounds having a quaternary ammonium group are describedin WO2014/011047, including a description of synthesis in the examples.

The compounds of the present invention are unexpectedly active againstchronic obstructive airway diseases such as COPD or asthma.

The compounds according to the present invention preferably have aTrolox oxidation equivalent, which is comparable or less than trolox,but their activity in preventing cell-damage is substantially improved.

Considering that the present compounds target the lungs, inhalation isthe most preferred administration route to be used in the presenttreatment of chronic obstructive airway diseases, such as chronicobstructive pulmonary disease (COPD) or asthma or bronchiectasis, andespecially chronic obstructive pulmonary disease (COPD). Inhaledcompounds can be absorbed quickly, and can act both locally andsystemically. Because proper techniques with inhaler devices innecessary to achieve the correct dose, the present invention, accordingto a further aspect, relates drug delivery device wherein the device isan inhaler such as a nebulizer comprising the present compounds, andcomprising the active ingredient or a pharmaceutically acceptable saltor base thereof in a formulation suitable for inhalation.

An inhaler, or puffer, is a medical device used for deliveringmedication into the body via the lungs. An inhaler is generally used inthe treatment of asthma and Chronic Obstructive Pulmonary Disease(COPD). To reduce deposition in the mouth and throat, and to reduce theneed for precise synchronization of the start of inhalation withactuation of the device, MDIs are sometimes used with a complementaryspacer or holding chamber device. Types of inhalers are metered-doseinhalers, dry powder inhalers and nebulizers.

The most common type of inhaler is the pressurized metered-dose inhaler(MDI). In MDIs, medication is most commonly stored in solution in apressurized canister that contains a propellant, although it may also bea suspension. The MDI canister is attached to a plastic, hand-operatedactuator. On Activation, the metered-dose inhaler releases a fixed doseof medication in aerosol form. The aerosolized medication is drawn intothe lungs by continuing to inhale deeply before holding the breath forapproximately 10 seconds to allow the aerosol to settle onto the wallsof the bronchial and other airways of the lung.

Dry powder inhalers of DPI release a metered or device-measured dose ofpowdered medication that is inhaled through a DPI device. Nebulizerssupply the medication as an aerosol created from an aqueous formulation.

The compound according the invention is formulated such that it issuitable for inhalation. In a preferred embodiment, the aerodynamicdiameter of the drug is in the range of 0.5-8 μm, more preferably in the1-5 μm aerodynamic diameter range. In this range, the drug is mostefficiently absorbed, because it related to particle dynamic behaviorand describes the main mechanisms or aerosol deposition; bothgravitational settling and inertial impaction depend on aerodynamicdiameter. The formulation may further comprise excipients, although thisis not necessary. Suitable excipients include lactose, glucose andmannitol, of which lactose is preferred.

Preparing a drug for inhalation is known, as for example described inRespiratory Care (2005) 50: 1209-1227. For MDI, a propellant will bepresent, and optionally a surfactant.

The amount of compound according to the present invention to beadministered per actuation of an inhaler is about 1 mmol or less,preferably about 0.3 mmol or less. The molecular weight of the compoundbeing generally below 400 g/mol, this means that the amount to beadministered per actuation is about 200 mg or less, preferably about 100mg or less. Generally, the amount of compound according the presentinvention is 1 μmol or more, preferably about 10 μmol or more.Generally, the amount of compound will be about 100 μg or more.

The compound of the present invention can be combined with other knowntreatments of asthma or COPD, such as described above. In particular,the compound of the present invention may be combined withcorticosteroids and/or long-acting or short-acting β-agonists and/orleukotrienes. The combination therapy may be effected in the sameinhaler, or in multiple inhalers.

The present invention will be further illustrated using the examplesbelow. In the examples, reference is made to figures wherein

FIG. 1: shows that SUL-compounds do not alter cell viability. hTERTcells were incubated for 24 hours with the indicated concentrations ofSUL90, SUL121, SUL127 and SUL136 in the absence of presence of 15% CSE.The H2S donor NaSH (500 μM) served as control. Data are expressed asmean±SEM, n=4-5, *p<0.05 vs. control in one-way ANOVA followed byBenferroni post hoc test;

FIG. 2: shows that Sul-90 and Sul-121 inhibit CSE-induced IL-8 releasefrom hTERT cells, hTERT cells were incubated for 24 hours with theindicated concentrations of Sul-90 and Sul-121 in the absence ofpresence of 15% CSE. The ß2-agonist fenoterol (Feno, 1 μM) and the H₂Sdonor NaSH (500 μM) served as controls. Data are expressed as mean±SEM,n=4-5, *p<0.05 vs. control in one-way ANOVA followed by Benferroni posthoc test;

FIG. 3 shows that Sul-90 and Sul-121 induce relaxation ofmethacholine-pre-contracted BTSM strips. The upper panel illustrates theprotocol of the isometric tension measurements. BTSM strips werepre-contracted with 1×10−3.5 μM methacholine, followed by the additionof the indicated concentrations of Sul-compounds. DMSO (0.5%) served ascontrol. Graphs represent means±SEM of 6 experiments. *p<0.05 vs.control in two-ways ANOVA;

FIG. 4: shows that the ß2-adrenoceptor antagonist propranolol does notalter the relaxation of BTSM strips induced by Sul-90 and Sul-121. BTSMstrips were pre-contracted with 1×10−3.5 μM methacholine, followed bythe addition of Sul-90 and Sul-121 (30 μM each) in the presence andabsence of 1 μM propranolol. Graphs represent means±SEM of 3experiments. *p<0.05 vs. control in two-way ANOVA;

FIG. 5: shows that Sul-121, but not Sul-90, shifts the dose responsecurve for isoprenaline to the right. BTSM strips were pre-contractedwith 1×10×3.5 μmethacholine, followed by the addition of Sul-90 andSul-121 (30 μM each) followed by a dose response curve for isoprenaline.DMSO (0.03%) served as control. Graphs represent means±SEM of 3experiments. *p<0.05 vs. control in two-way ANOVA;

FIG. 6: shows that Sul-121 and Sul-90 decrease the contraction inducedby methacholine, BTSM strips were pre-incubated with Sul-90 and Sul-121(30 μM each), followed by a dose response curve for methacholine. DMSO(0.03%) served as control. Graphs represent means±SEM of 3 experiments.*p<0.05 vs. control in two-way ANOVA.

FIG. 7 shows that experiments in guinea pigs have been, performed asdescribed in Example 3. FIG. 7 shows the effect of Sul-121 on airwayhyperresponsiveness after LPS challenging.

FIG. 8 shows the effect of Sul-121 on inflammatory cells in a guinea pigmodel after LPS challenging.

EXAMPLES Example 1: Synthesis of Several Compounds

Compounds according to the invention can be synthesized according tostandard synthesis methods which are well known by a person skilled inthe art. SUL-0083, SUL-0084 and SUL-0085 are commercially available.Table 1 below provides a summary of the present compounds as aninterchangeable arbitrary indication (code) of the present compoundsused herein.

TABLE 1 Several compounds according to the present invention CodeChemical name SUL-083 2,2,5,7,8-pentamethylchroman-6-ol SUL-084(S)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid SUL-085(R)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid SUL-0896-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide SUL-090N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-091N-butyl-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-0926-hydroxy-N-isopropyl-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-093(E)-N-(3,7-dimethylocta-2,6-dien-1-yl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-095(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(morpholino)methanone;SUL-097N-(4-fluorobenzyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;SUL-0986-hydroxy-N-((S)-2-hydroxy-1-phenylethyl)-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-1006-hydroxy-2,5,7,8-tetramethyl-N-(2-(methylamino)ethyl)chroman-2-carboxamide; SUL-1016-hydroxy-N,2,5,7,8-pentamethyl-N-(2-(methylamino)ethyl)chroman-2-carboxamide; SUL-1026-hydroxy-2,5,7,8-tetramethyl-N-(3-(piperidin-1-yl)propyl)chroman-2-carboxamide; SUL-1046-hydroxy-2,5,7,8-tetramethyl-N-(3-nitrophenyl)chroman-2-carboxamide;SUL-106N-(4-fluorophenyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;SUL-107 methyl4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamido)benzoate; SUL-108(4-butylpiperazin-1-yl)(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)methanone; SUL-109(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone; SUL-110((2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl)(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)methanone; SUL-111N-((R)-2-amino-2-oxo-1-phenylethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-112(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone; SUL-114N-(2-bromoethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;SUL-115N′-(2-cyanoethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carbohydrazide;SUL-1162-(((4-fluorobenzyl)amino)methyl)-2,5,7,8-tetramethylchroman-6-ol;SUL-117 2-((butylamino)methyl)-2,5,7,8-tetramethylchroman-6-ol; SUL-1186-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylic acid; SUL-1192-(hydroxymethyl)-5,7-diisopropyl-2,8-dimethylchroman-6-ol; SUL-1206-hydroxy-N-((R)-1-hydroxypropan-2-yl)-2,5,7,8-tetramethylchroman-2-carboxamide SUL-121(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanoneSUL-122 (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-(2-hydroxyethoxy)ethyl)piperazin-1-yl)methanone; SUL-123N-(2-cyanoethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;SUL-124 6-hydroxy-N-(2-((2-hydroxyethyl)(methyl)amino)ethyl)-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-125(R)-N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-126(S)-N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-1282-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol; SUL-1292-((((S)-2-hydroxy-1-phenylethyl)amino)methyl)-2,5,7,8-tetramethylchroman-6-ol; SUL-1302,5,7,8-tetramethyl-2-(piperidin-1-ylmethyl)chroman-6-ol; SUL-131N,6-dihydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxamide; SUL-132(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone; SUL-133(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone; SUL-1342-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol; SUL-1352-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol; SUL-1362-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetic acid; SUL-137(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-yl)(piperazin-1-yl)methanone; SUL 138(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone; SUL-1392-(4-(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carbonyl)piperazin-1-yl)acetic acid; SUL-140 ethyl2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetate; SUL-141(S)-2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetic acid; SUL-142(R)-2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetic acid; SUL-143(2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylic acid; SUL-144(2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylic acid; SUL-145(2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylic acid;Synthesis of SUL 089-112, 114-117, 120-126, 128-130, 132, 134-135, 138,and 140

Amidation of trolox was achieved by reaction with the appropriate aminein the presence of standard coupling reagents for amide formation, e.g.,HATU and CDI. The corresponding amines were prepared by reduction of theamides formed with BH₃ Hydroxamic acid derivatives were prepared byreaction with hydroxylamine/CDI. The synthesis of carbohydrazideanalogues of trolox was achieved by reaction with (substituted)hydrazines. Enantiomeric/diastereomeric compounds were prepared startingfrom enantiomerically pure (R)- or (S)-Trolox or by means of chiralchromatography.

Synthesis SUL-118, SUL-119 en SUL-146

Oxidation of commercially available propofol with salcomine, acoordination complex of the salen ligand with cobalt, followed byreduction with NaBH₄ afforded 2,6-diisopropylbenzene-1,4-diol Subsequentmethylation with HCO/SnCl₂/HCl and reaction with methyl methacrylatefurnished SUL-146 (methyl6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylate). Hydrolysiswith LiOH yielded the carboxylic acid SUL-118(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylic acid). Thealcohol SUL-119(2-hydroxymethyl)-5,7-disopropyl-2,8-dimethylchroman-6-ol) was obtainedby reduction of SUL-146 with LiAlH₄.

Synthesis of SUL-131, SUL-133, SUL 137 en SUL-146

Starting from the carboxylic acid SUL-118(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylic acid), thehydroxylamine was obtained by reaction with hydroxylamine using CDI ascoupling reagent. Compounds SUL 122((6-hydroxyl-5,7-diisopropyl-2,8-dimethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone) were prepared by reactionof SUL-118 with the appropriate piperazine derivative. Both couplingreagents HATU and CDI resulted in satisfactory yields. SUL 139(2-(4-(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carbonyl)piperazin-1-yl)aceticacid) was prepared by a reductive amination of SUL 137((6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-yl(piperazin-1-yl)methanone)with glyoxalic acid.

Synthesis of SUL-136, SUL-141 and SUL-142

Hydrolysis of SUL-140 (ethyl2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetateunder N₂ atmosphere furnished SUL-136(2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)aceticacid) in high yield. The enantiomers SUL-141 and SUL-142 were preparedaccording to the above-described conditions.

Synthesis of SUL 143, 144 en 145

Amidation of trolox with (S)-methyl pyrrolidine-2-carboxylate (L-prolinemethyl ester) afforded, after column chromatography, twodiastereoisomers. Subsequent hydrolysis of the individualdiastereoisomers afforded SUL-144((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylicacid, diastereomer 1) and SUL-145((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylicacid, diastereomer 2). The racemic analogue SUL-143((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylicacid) was obtained by mixing the esters of the individualdiastereoisomers followed by hydrolysis of the ester moiety using LiOH.

Amidation of Trolox (General Example) SUL-108((4-butylpiperazin-1-yl)(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)methanone.HCl

Trolox (11 g, 0.044 mol, 1 eq.) was suspended in acetonitrile (100-150ml). CDI (8.6 g, 0.053 mol, 1.2 eq.) was added in portions. The reactionmixture was stirred for 0.5-1 hour at room temperature. After additionof 1-butylpiperazine (6.9 g, 0.048 mil, 1.1 eq.) the reaction mixturewas stirred at 25-30° C. over the weekend. The reaction mixture wasconcentrated, H₂O (200 ml) was added and the aqueous layer was extractedwith EtOAc (4×). The combined organic layers were dried, filtered andconcentrated. The crude product obtained was purified by columnchromatography (DCM/10% MeOH) affording the compound aimed for (9 gproduct, 82% pure). Crystallization from EtOAc/heptanes afforded SUL-108(6 g, 0.016 mol, 36% yield, 90% pure) as a white solid. The materialobtained was dissolved in DCM (50-100 ml). HCl (4 M in dioxane, 8.8 ml,0.0035 mol, 2.2 eq.) was added and the reaction mixture was stirred atroom temperature over the weekend. The mixture was filtered, rinsed withDCM, and dried to afford the HCl sale of SUL-108 (6.3 g, 97-98% pure) asa white solid.

¹H-NMR (CDCl₃, in ppm): 0.93 (t, 3H), 1.38 (m, 2H), 1.58 (s, 3H), 1.67(m, 2H), 2.09 (s, 3H), 2.12 (s, 3H), 2.15 (s, 3H), 2.50-3.20 (m, 14H),M⁺=375.3

Reduction of Trolox Amides (General Example) SUL-128.(2-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol.HCl

BH₃, THF in THF (16 ml, 0.0156 mol, 2 eq.) was cooled to T=(0° C. Asolution of SUL-112((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)((S)-2-(hydroxymethyl)pyrrolidin-1-yl-methanone;2.6 g, 0.0078 mol, 1 eq.) in THF (50 ml) was added drop-wise and thereaction mixture was refluxed for 1 hour and cooled to room temperatureovernight. The reaction mixture was cooled on an ice bath and HCl (6 M,25 ml) was added drop-wise. DCM (100 ml) was added and the layers wereseparated. The aqueous layer was extracted with DCM (3×). The combinedorg. layers were dried over K₂CO₃ until no gas formation was noticedanymore. The organic phase was filtered and concentrated. The crudeproduct was cooler on an ice bath, and NaOH (6M, 50 ml) was addeddrop-wise. After addition the reaction mixture was stirred for 1 hourand extracted with DCM (4×). The combined DCM layers were dried,filtered and concentrated to give 1.6 g crude product (20-40% pure). Thematerial was purified by column chromatography affording SUL-128 (300mg, 0.94 mmol, 12% yield, 90% pure). This was dissolved in DCM (10 ml)and cooled to T=0° C. (ice bath). HCl (4M in dioxane, 0.3 ml, 0.94 mmol,1.2 eq.) was added and the reaction mixture was stirred at roomtemperature overnight. The solid formed was filtered, washed with Et₂Oand dried to afford the HCl salt of SUL-128 (300 mg, 90% pure) as awhite solid (mixture of diastereomers).

¹H-NMR (CDCl₃, in ppm): 1.20-1.90 (m, 7H), 2.12 (s, 6H), 2.17 (s, 3H),2.20-2.90 (m, 9H), 3.4-3.65 (m, 2H), M⁺=320.1

Synthesis of SUL-118(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylic Acid)Synthesis of 2,6-Diisopropropylcyclohexa-2,5-diene-1,4-dione

Propofol 100 g, 561 mmol) was dissolved in DMF (250 mL). The solutionwas cooled to 0° C. while stirring. Salcomine (16.6 g, 51 mmol; 9 mol %)was added and the resulting reaction mixture was stirred 112 h overnightwhile warming to room temperature. The reaction mixture was poured inwater (7 L). The resulting slurry was extracted with heptanes (5×1 L).The combined organic extracts were dried with Na₂SO₄Concentration of thesolution under vacuum afforded the crude2,6-diisopropylcyclohexa-2,5-diene-1,4-dione (62.5 g; 325 mmol; 58%yield) as an oil. The product was used in the next step without furtherpurification.

Synthesis of 2,6-Diisopropylbenzene-1,4-dio

Crude 2,6-diisopropylcyclohexa-2,5-diene-1,4-dione (62.5 g, 325 mmol)was dissolved in dichloromethane (300 mL) and methanol (100 mL). Thesolution was cooled to 0° C. with an ice bath. Sodium borohydride (4.5g, 182 mmol) was added in portions. After the addition was complete thereaction mixture was stirred at room temperature overnight. Acetone (150mL) was added to quench the excess of sodium borohydride. After 30minutes stirring 2N aq. HCl (200 mL) was added. After stirring for 45minutes the mixture was extracted with ethyl acetate (4×400 mL). Thecombined organic layers were dried with Na₂SO₄. Concentration of thesolution under vacuum afforded crude 2,6-diisopropylbenzene-1,4-diol (64g, 330 mmol) as a red oil in quantitative yield. The product was used isthe next step without further purification.

Synthesis of 3,5-Diisopropyl-2-methylbenzene-1,4-diol

A mixture of 2,6-diisopropylbenzene-1,4-diol (64 g, 0.33 mol),paraformaldehyde (9.8 g, 0.327 mol), SnCl₂ (217.9 g, 1.15 mol),concentrated aq. 37% HCl (0.6 L) and diisopropyl ether (2.5 L) washeated to reflux for 4 hours. After cooling to room temperatureovernight the biphasic mixture was separated. The aqueous layer wasextracted with TBME (2000 mL). The combined organic fractions werewashed with 1N aq. HCl (1000 mL), water (1000 mL) and brine (1000 mL).The organic fractions were dried with Na₂SO₄ and concentrated undervacuum to give a 50:35 mixture of3,5-diisopropyl-2-methylbenzene-1,4-diol and2,6-diisopropyl-3,5-dimethylbenzene-1,4-diol (61 g oil) according toGCMS analysis. Purification by chromatography on silica gel (1200 mL)eluting with ethyl acetate/heptanes=97.5:2.5 (4000 mL), 95:5 (4000 mL)gave 3,5-diisopropyl-2-methylbenzene-1,4-diol 6 (16.6 g, 79.8 mmol; 24%:83% pure) as an oil.

Synthesis of Methyl6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylate

3,5-diisopropyl-2-methylbenzene-1,4-diol (10.6 g, 50.9 mmol; 83% pure)was dissolved in methyl methacrylate (20 mL, 186 mmol). The solution wastransferred to a Teflon tube in a Berghof reactor. Aqueous formaldehyde(10 mL; 37% wt. solution, stabilized with 10-15% MeOH) was added and thereaction mixture was heated to 180° C. (internal temperature) in theclosed reactor for 5 hours while stirring. After cooling to ca. 40° C.the reaction mixture was poured in MeOH (200 mL) and the mixture wasconcentrated under vacuum. Purification by chromatography on silica gel(600 mL) eluting with ethyl acetate/heptanes=95:5 (5000 mL; TLC: Rf˜0.2;spot stained with iodine vapor) gave the desired pure product methyl6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylate (10.0 g,31.3 mmol, 61%).

Synthesis of 6-Hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylicAcid (SUL-118)

A mixture of purified methyl6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylate (8.3 g, 25.9mmol) and lithium hydroxide monohydrate (4.3 g, 102.5 mmol; 4 eq.) inMeOH (100 mL). THF (100 mL) and water (25 mL) was heated for 30 minutesat ambient pressure while rotating with a rotary evaporator in a warmwater bath at 60° C. The organic solvents were evaporated under vacuum.Water (150 mL) was added, to the residue, followed by acetic acid. (10mL). A light orange mixture was obtained. Extraction with ethyl acetate(3×100 mL), drying of the combined organic fractions with Na₂SO₄ andconcentration under vacuum gave the crude product as an orange solid.The solids were stirred with tBME (150 mL). A beige solid precipitatedand an orange solution was obtained. Heptane (250 mL) was added and themixture was stirred for 15 minutes. The mixture was filtered over aglass filler. The residual solids were washed with heptanes (2×50 mL) onthe filter under section. Drying of the solids under vacuum at 60° C.gave pure 6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylicacid (SUL-118) as an off-white solid (3.1 g, 10.13 mmol; 39%, 100%pure).

¹H-NMR (CDCl₃, in ppm): 1.38 (t, 12H), 1.52 (s, 3H), 1.87 (m, 1H), 2.20(s, 3H), 2.30 (m, 1H), 3.20 (m, 1H), 3.38 (m, 1H). M+=307.10

Synthesis of SUL 119(2-(hydroxymethyl)-5,7-diisopropyl-2,8-dimethylchroman-6-ol)

A solution of methyl6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylate (500 mg,1.56 mmol) in THF (12 mL) was added over 5 minutes with a syringe via arubber septum to LiAlH₄ (238 mg, 6.26 mmol; 4 eq.), pre-weighted in adry 3-mecked 100 mL roundbottomed flask under inert nitrogen atmospherewhile stirring at room temperature. The exothermic addition of the esterwas accompanied with gas evolution. After the addition was complete theresulting grey suspension was heated to reflux. After 3 hours theheating was stopped and the reaction was quenched by dropwise additionof EtOAc (6 mL; exothermic). Water (5 mL) was added in small portions,followed by 2N HCl (2 mL) followed by EtOAc (25 mL). The mixture waspoured on Na₂SO₄ (ca. 50 g) and the slightly yellow organic layer wasseparated from the two-phase mixture. The aqueous phase was washed withEtOAc (50 mL) and the combined organic fractions were concentrated undervacuum to give the crude alcohol (530 mg) as a clear oil. Heptane (100mL) was added and after concentration under vacuum the2-(hydroxymethyl)-5,7-diisopropyl-2,8-dimethylchroman-6-ol (248 mg, 0.85mmol, 54%, LCMS; 95.5% pure).

M+=293.2

Synthesis of SUL 139(2-(4-(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carbonyl)piperazin-1-yl)aceticAcid

SUL-137 (440 mg, 1.17 mmol, 1 eq.) was dissolved in MeOH (50 ml) andglyoxalic acid (216 mg, 2.35 mmol, 2 eq.) was added. The resultingmixture was stirred for 1 hour at room temperature and, subsequently.NaBH₃CN (183 mg, 2.94 mmol, 2.5 eq.) was added. The reaction mixture wasstirred at room temperature overnight. Acetic acid (few ml) was addedand after stirring at room temperature for 0.5-1 hour, the reactionmixture was concentrated. The residue obtained was dissolved in EtOAc,washed with H₂O (2×), dried filtered and concentrated to afford SUL-139(500 mg, 1.16 mmol, 98%, 91-92% pure) as a light yellow solid.

¹H-NMR (CD₃OD, in ppm); 1.33 (dd, 12H), 1.59 (s, 3H), 1.62 (m, 1H), 2.09(s, 3H), 2-5-3.0 (m, 7H), 3.1-3.6 (m, 4H), 3.81 (bs, 2H), 4.28 (bs, 2H).M⁺=433.2.

Synthesis of SUL 136(2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)aceticAcid

A 250 ml three-necked flask equipped with two septa (left and right) anda stopcock was charged with SUL-136 (15.5 g, 38.4 mmol) and THF/water(240 ml THF+80 ml water). The clear solution was stirred and degassedfor at least 30 minutes by argon-bubbling, using an inlet tube equippedwith a long syringe needle through the left septum; the right septum wasequipped with a short needle and functioned as outlet. The degassedsolution (which was maintained under argon) was cooled to 0° C. in anice-bath and solid anhydrous LiOH (2.3 g, 96 mol, 2.5 eq.) was added inone portion. The resulting reaction mixture was stirred for 2 hours at0° C. after which it was neutralized by addition of a MeOH/water (3/1,v/v) slurry of Dowex-50WX8-200 ion-exchange resin; the final pH wasapprox. 6. The Dowex resin was filtered off with suction and rinsed with3 portions of MeOH/water (3/1, v/v). The filtrate was reduced in vacuoand to the wet product was added approx. 100 ml water. The resultingwhite aqueous suspension was freeze-dried overnight to afford SUL-136(13.48 g, 93%, LCMS; 99.6%) as a white solid.

1H-NMR (CD3OD, in ppm)): 1.60 (s, 3H), 1.65 (m, 1H), 2.05 (s, 3H), 2.10(s, 6H), 2.55 (m, 2H), 2.62 (m, 1H), 3.0, (bs, 4H), 3.40 (bs, 2H), 3.65(bs, 2H), 4.25 (bs, 2H). M+=377.1

Synthesis of SUL 144((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylicAcid)

(2S)-methyl1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylate(diastereomer 1, 3.5 g, 9.7 mmol) was dissolved in THF/H₂O (60/20 mL).N₂ was bubbled through the solution for 1 h. The mixture was cooled inan ice-bath and LiOH.H₂O (1.01 g, 24.2 mmol, 2.5 eq.) was added. Thereaction mixture was stirred under N₂ at RT overnight. Dowex-50WX8-200(washed 4× with MeOH/H₂O 3:1) was added as a slurry in MeOH/H₂O (3:1)until the pH=6. The mixture was filtered, washed with MeOH/H₂O (3:1) andconcentrated in vacuo. Demi H₂O (50 mL) was added to the concentrate andthe solution was freeze dried affording SUL-144 (3.4 g, 9.7 mmol, quant,99.7% pure) as a off-white foam.

1H-NMR (CDCl3): 1.60 (s, 3H), 1.65-2.30 (m, 14H), 2.60 (m, 2H), 2.81 (m,1H), 3.49 (m, 1H), 4.01 (t, 1H), 4.50 (d, 1H), M+=348.1

Example 2

Introduction

H₂S alters biological functions through the interplay of severaldistinct signaling mechanisms. Using CTH knocks-out mice, the role ofH₂S was studied in airway hyperreponsiveness (AHR) and inflammation in amouse of asthma. It was reported that the expression of CTH andendogenous H₂S production was reduced in lungs of CTH-deficient micecompared to wild-type mice. Administration of ovalbumin to induce acuteasthma reduced the CTH expression and H₂S production in wild-type mice.Depletion of CTH lead to an increased AHR, airway inflammation, andelevated levels of IL-5, IL-13 and eotaxin-1 in bronchoalveolar fluidafter ovalbumin challenge, features being reversed upon treatment withthe H₂S donor NaHS These findings clearly indicate that the CTH/H2Ssystem plays a critical protective role in the development of asthma.

Intriguingly, there is a strong relationship between sputum and H₂Slevels for patients with severe asthma. Sputum H₂S level represents anovel promising biomarker for obstructive lung disease such as asthma,neutrophilic inflammation, chronic airflow obstruction and also as areflection of ß-adrenergic bronchodilator responsiveness. It has beenproposed that the combined use of the ß-agonist fenoterol and H₂Smeasurement might offer a more comprehensive description of obstructivelung disease phenotypes.

In a rat model of hypoxia-induce pulmonary vascular structural changes,the H₂S donor NaHS reduced the expression of the remodeling parametercollagen I, collagen III and transforming growth factor-ß (TGF-ß) andinhibited the proliferation of pulmonary artery smooth muscle cells.Although current studies are not yet direct related to human asthma, itis well established that the severity of asthma is worsened through anincrease of airway smooth muscle mass, it is tempting to assume thatTGF-ß further promotes the increase in airway smooth muscle mass. Adecrease in the level of TGF-ß by H₂S may effectively protect againstprocesses underlying airway remodeling.

Mouse models of acute lung injury induced by combined burn and smokeinhalation, have shown that post-treatment administration of the H₂Sdonor NaHS-decreased mortality and increased median survival in mice.H₂S also inhibited the level of IL-1ß, but enhanced the level of theanti-inflammatory cytokine IL-10. It generally assumed that IL-10 exertsprotective biological functions by suppressing the expression ofadhesion molecules as well as reducing the level of macrophages andneutrophils, processes most likely involving the inhibition of thepro-inflammatory transcription factor NF-kB. Additionally, it has beenproven that IL-1ß exerts pro-inflammatory effects on the airway mucosaltissue. Thus, it is reasonable to propose that H₂S exerts protectiveeffects in acute long injury through alterations in the balance of thepro-inflammatory IL-1ß and the anti-inflammatory IL-10.

As outlined above, several recent disclosures indicate that H₂S is ofcentral importance in the regulation of biological functions throughoutthe human body. H₂S dysfunction under pathophysiological circumstancesof chronic obstructive pulmonary diseases, such as asthma and COPD,contributes to the progression of disease symptoms both in animal modelsand patients.

In this example, the effects of four H₂S compounds, i.e. SUL90, SUL121were studied on:

1) The cell viability of human (immortalized) airway smooth muscle cells(hTERT cells),

2) The release of the inflammation mediator IL-8 from hTERT cells,

3) Airway smooth muscle contractility of bovine trachea smooth musclestrips.

These samples used were:

-   -   Two SUL-compounds: SUL90, SUL121;    -   Human telomerase reverse transcriptase immortalized airway        smooth muscles (hTERT) cells, cultured as previously described        (Oldenburger et al., 2012). Prior to the experiments, cells were        serum-deprived, for 1 day, followed by cell treatment with the        indicated concentrations of the SUL-compounds in the absence and        presence of 15% cigarette smoke extract (CSE) for additional 24        hours. As controls, 1 μM fenoterol and 500 μM of the H2S donor        NaHS were used,    -   100% cigarette smoke extract (CSE), freshly made by combusting        (Watson Marlow 323 E/D, Rotterdam, The Netherlands) the smoke of        two research cigarettes (University of Kentucky 2R4F) through 25        mL of DMEM (FBS free) at a speed of approximately 1 cigarette/5        minutes. Afterwards. CSE was diluted to 15% (Oldenburger et al.,        2012).

For cell-based studies, the SUL compounds were dissolved in 0.9% NaCl as1 mM stock solutions. For the isometric tension measurements, the SULcompounds were dissolved in 100% DMSO as 100 mM stock solutions.

Assay 1: Trypan Blue Cell Counting

For cell viability measurements, trypan blue cell counting was performedas previously described (Oldenburger et al., 2012). As control, 500 μMof the H₂S donor NaHS was used. Alternatively, alamar blue measurementswere performed to determine cell viability essentially as describedbefore (Oldenburger et al., 2012).

Briefly, hTERT cells were plated on 24-well plates as a cell density of10,000 cells/well. Again cells were serum-deprived for 1 day, followedby cell treatment with the indicated concentrations of the SUL-compoundsin the absence and presence of 15% cigarette smoke extract (CSE) foradditional 24 hours

Assay 2: Release of Interleukin-8 (IL-8) from hTERT Cells

This assay was used to determine the release of interleukin-8 from hTERTcells, fenoterol (1 μM) and the H₂S donor (500 μM) served as controls.24 hours after cell stimulation with the indicated concentrations of theSUL-compounds in the absence and presence of 15% CSE, culture medium wascollected to measure the IL-8 concentration in the cell supernatantsaccording to the manufacturer's instructions (Pelikine Compact ELISAkit, Sanquin, The Netherlands), as previously described (Oldenburger etal., 2012.

Assay 3: Bovine Trachea Smooth Muscle (BTSM) Strips and IsometricTension Measurements

Isometric tension measurements were performed as described previously(Roscioni et al., 2011; Roscioni, Prins et al., 2011). BTSM strips weremounted for isometric recording in organ-baths, containingKrebs-Henseleit (KH) buffer, containing in mM: 117.5 NaCl, 25 NaHCO₃,5.5 glucose, 5.6 KCl, 1.18 MgSO₄, 2.50 CaCl₂, 1.28 NaH₂PO₄, pre-gasseswith 5% CO₂ and 95% O₂, pH 7.4. After dissection of the smooth musclelayer and careful removal of connective tissue, BTSM strips ofapproximately 1 cm length and 2 mm width were prepared. Tissue stripswere cultured in DMEM supplemented with non-essential amino acid mixture(1:100), sodium pyruvate (1 mM), gentamicin (45 μg*ml-1), penicillin(100 U*ml-1), streptomycin 100 μg*ml-1, amphotericin B (1.5 μg*ml-1)apo-transferrin (5 μg*ml-1) and ascorbic acid (100 μM). The BTSM stripswere cultured for 1-3 days before isometric tension measurement in anInnova 4000 incubator shaker 7° C., 55 rpm).

For isometric tension measurements (Roscioni et al., 2011; Roscioni,Prins et al., 2011), BTSM strips were calibrated, were mounted into thetransducers and submerged into the organ baths in pre-gassed KH buffer.Each strip was adjusted to a basal tension of 3 gram. Then, the stripswere washed, equilibrated again for 60 minutes, followed bypre-contractions induced by 1×10×3.5 μM methacholine. To analyze acuteeffects of the SUL-compounds on the isometric tension, the strips wereincubated with accumulative doses of the SUL-compounds (1-300 μM),followed by the addition of 0.01 μM isoprenaline.

To analyse a potential role of the ß2-AR in the effects induced by theSUL-compounds, the strips were incubated with 1 μM propranolol for 30minutes prior addition of the SUL-compounds. To analyze potentialeffects of the SUL-compounds on the isoprenaline-induced relaxation, thestrips were first incubated with the SUL-compounds (30 μM each),followed by the addition of accumulative doses of isoprenaline (1×10−5-1μM). Finally, to analyze potential effects of the SUL-compounds on themethacholine-induces contraction, the strips were first incubated withthe SUL-compounds (30 μM each), followed by addition of accumulativedoses of methacholine (0.0001-30 μM), before the addition of 0.01 μMisoprenaline.

Data are shown as mean±standard error of the mean. One-way ANOVAfollowed by Bonferroni post hoc test, 2-tailed paired t-test, two-wayANOVA was used when appropriate to identify statistical differencesbetween means. A statistical difference was defined as significant atp<0.05.

Results

SUL-Compounds on Cell Viability

As illustrated in FIG. 1, the SUL-compounds exert no significant effecton cell viability. Increasing concentrations of the SUL-compounds,however, seem to further increase the profound effect of CSE on cellviability. Shown here are cell viability studies based on trypan bluecounting. Similar results were obtained using alamar blue measurements(data not shown). Thus SUL-90 and SUL-121 seem not to severely after thecell viability of hTERT cells.

The Effect of Sul-90, Sul-121 on the Release of IL-8 from hTERT Exposedto CSE

As illustrated in FIG. 2, the Sul-compounds exert differential effectson the cellular release of IL-8 induced by CSE. Sul-90 and Sul-121significantly reduce the release of the inflammatory mediator IL-8 (FIG.5).

The Effects of Sul-90, Sul-121, Sul-127 and Sul-136 on the AcuteRelaxation of BTSM Strips

As illustrated in FIG. 3, Sul-90 shows a trend to induce relaxation ofBTSM strips at concentration higher than 100 μM. Sul-121 induces evenmore pronounced relaxation, reaching statistical significance. Incontrast, Sul-127 and Sul-136 do not alter the contractile tone of BTSMstrips (FIG. 3).

The Relaxation Induced by Sul-90 and Sul-121

To Analyze a potential involvement of the ß2-adrenoceptor in theirrelaxing properties, the BTSM strips were pre-incubated with theß2-adrenoceptor antagonist propranolol. As illustrated in FIG. 4,propranolol induced a right-ward shift of the dose-response curve forisoprenaline. In contrast, relaxation induced by Sul90 and Sul-121 wasnot affected by propranolol (FIG. 4). In the presence of propranolol,Sul-90 even showed a trend to a left-ward shift of its relaxingproperties. Statistical analysis (see Table 2) revealed that propranololsignificantly altered the relaxation by isoprenaline, but left therelaxation induced by Sul-90 and Sul-121 unaffected. Thus, Sul90 andSul-121 induce acute relaxation of BTSM independent of theß2-adrenoceptor.

TABLE 2 Statistical anaylsis: pD2 values were calculated from individualexperimental data. Each value represented the mean ± SEM from 3determinations. Statistical analyses were performed by a one-way ANOVA.p < 0.001 vs. all other agonists; p < 0.001 vs. solvent treated bovinetracheal smooth muscle tissue Agonist Solvent Propranolol DMSO 3.6 ± 0.13.6 ± 0.2 Sul-90 3.4 ± 0.0 3.5 ± 0.0 Sul-121 3.5 ± 0.0 3.5 ± 0.1Isoprenaline 7.7 ± 0.2 5.4 ± 0.1The Impact of Sul-90 and Sul-121 on the Isoprenaline-Induced Relaxation

The BTSM strips were pre-incubated with Sul90 and Sul-121 at aconcentration of 30 μM shown before to leave the isometric tensionunaffected. As illustrated in FIG. 5, Sul-121, but not Sul-90, induced asignificant right-ward shift of the dose response curve forisoprenaline.

The Impact of Sul-90 and Sul-121 on the Methacholine-Induced Contraction

BTSM strips were pre-incubated with Sul90 and Sul-121 at a concentrationof 30 μM shown before to leave the isometric tension unaffected. Asillustrated in FIG. 6, Sul-90 and Sul121 reduced the contraction inducedby methacholine.

Conclusions

-   1) SUL90 and SUL121 do not severely alter cell viability of hTERT    cells.-   2) SUL90 and SUL121 inhibit the cellular IL-8 release induced by    CSE.-   3) SUL90 and SUL121 induce relaxation of bovine trachea strips    pre-contracted with methacholine in a ß2-adrenoceptor-independent    manner.-   4) SUL212 induces a right-ward shift of the dose response curve for    isoprenaline indicating that SUL121 may compete for intracellular    signaling components of isoprenaline.-   5) SUL90 and SUL12 significantly reduce the contraction induced by    methacholine.

Example 3

Guinea pigs were instrumental with an intrapleural balloon catheterimplanted for online measurement of pleural pressure. 24 hours beforeLPS instillation (t=−24 h), the basal airway responsiveness to histamineis measured (PC100: histamine concentration inducing a doubling ofpleural pressure). 30 minutes prior to intranasal LPS instillation(t=−0.5 h), the animals were treated with saline,(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanone orN,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide or fenoterol,used as a positive control.

At time point 0 (t=0 h), LPS was instilled intranasally, after whichairway hyperreponsiveness was measured at different time point (t=1, 2,3, 6 and 24 h), by performing PC100 measurements. At t=25 h abronchoalveolar lavage (BAL) was preformed to determine the effects ofthe different treatments on airway inflammation. As a control for theLPS-induced effects, an intranasal challenge with saline was performedafter the saline treatment at t=0.5 h.

To assess effective doses, histamine PC100 measurements 30 min beforeand at various time points (30 min, 1 h, 2 h, 3 h, 6 h and 24 h) aftertreatment with either(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide were performs.Aerosol concentrations of 3, 30 and 300 mM were used for both compounds.

Under complete anesthesia, an intrapleural ballooncatheter wassurgically implanted in the pleural cavity for online measurement ofpleural pressure in freely moving animals. After one week of recovery,the animals were trained to adapt to the measuring method.

FIG. 7 shows the effect of a compound of the present invention on airwayresponsiveness, and the results show that the compound has an apparentdiletating effect.

FIG. 8 shows results of the BAL measurements, and—although the errormargin in the control is relatively large, the results indicate thateosinophils, lymphocytes, neutrophils and epithelial cells were allreduced. Thereby, this experiment shows that the compounds of thepresent invention have a reductive effect on the inflammation in vivo.

The invention claimed is:
 1. A method of treating a chronic obstructiveairway disease in a subject, the method comprising administering to thesubject a compound according to formula (I) or a pharmaceuticallyacceptable salt thereof,

wherein R1 and R2 are the same or different, and represent a C1-C4linear or branched alkylgroup; wherein R3 represents hydrogen; n is 1;R4 is CO—N—R5; wherein R5 comprises linear or branched alkyl groupcomprising 1-12 carbon atoms or cyclic alkyl group, substituted withnitrogen or oxygen; and wherein nitrogen is amine, quaternary amine,guanidine or imine, and oxygen is hydroxyl, carbonyl or carboxylic acid,and wherein oxygen and nitrogen together form amide, urea or carbamategroups; wherein the compound is in a formulation suitable forinhalation.
 2. The method of treating a chronic obstructive airwaydisease according to claim 1, wherein the compound according to formula(I) has a molecular weight lower than 500 Da.
 3. The method of treatinga chronic obstructive airway disease according to claim 1, wherein thecompound according to formula (I) does not comprise an aromaticheterocycle ring.
 4. The method of treating a chronic obstructive airwaydisease according to claim 1, wherein R5 is cyclic alkyl group.
 5. Themethod of treating a chronic obstructive airway disease according toclaim 1, wherein the compound is in solid form and has an aerodynamicdiameter of 0.5-8 μm.
 6. The method of treating a chronic obstructiveairway disease in a subject according to claim 1, wherein the molecularweight of R4 is less than 300 Da.
 7. The method of treating a chronicobstructive airway disease according to claim 1, wherein the compound isin solid form and has an aerodynamic diameter in the 1-5 μm aerodynamicdiameter range.
 8. A method of treating a chronic obstructive airwaydisease in a subject, the method comprising administering to the subjecta compound according to formula (I) or a pharmaceutically acceptablesalt thereof:

wherein R1 and R2 are methyl; wherein R3 represents hydrogen; n is 1;wherein the molecular weight of R4 is less than 300 Da; wherein R4 isCO—N—R5; wherein R5 is a cyclic alkyl group substituted with nitrogen,wherein nitrogen is amine, quaternary amine, guanidine or imine; andwherein the compound according to formula (I) has a molecular weightlower than 500 Da.